Mammalian cells tightly regulate their cholesterol content and distribution. In the liver, the movement of cellular cholesterol to the endoplasmic reticulum (ER) is critical for its conversion to cholesteryl esters and subsequent incorporation into nascent lipoproteins. It is also essential for metabolism of hepatic cholesterol to bile acids. The route and mechanism of cholesterol transport to the ER is unknown. Elucidation of cellular factors responsible for cholesterol movement to the ER may reveal new therapeutic targets for hypercholesterolemia. We have isolated a somatic cell mutant with defective trafficking of plasma membrane cholesterol to the ER (3,4). Chinese hamster ovary (CHO) mutant 3-6 transports both newly synthesized and lipoprotein-derived cholesterol to the plasma membrane, but fails to mobilize cell surface cholesterol to the ER for metabolism or regulation of homeostatic responses. This phenotype is likely due to a change in the plasma membrane lipid composition since, despite increased levels of cholesterol in the 3-6 plasma membranes, 3-6 cells are resistant to amphotericin B, and an polyene antibiotic that forms pores in cholesterol-rich membranes. Our hypothesis is that the 3-6 gene encodes a protein that affects the lipid organization of the plasma membrane. Loss of 3-6 activity alters the plasma membrane lipid domain structure such that cholesterol is both latent to amphotericin B and prevented from entering its endocytic pathway. The Aims of this study are: Specific Aim #1: To identify cDNAs that, when expressed in mutant 3-6 cells, correct the cholesterol transport defective phenotype. 3-6 cells expressing an ecotropic retroviral receptor will be transfected with a retroviral cDNA library, cDNAs that correct the phenotype will be identified. We will determine which of the correcting cDNAs encode the 3-6 protein. Specific Aim #2: To identify proteins whose expression levels are altered by the 3-6 mutation. Comparison of CHO vs. 3-6 proteomes and gene expression patterns by two-dimensional gel electrophoresis and microarray analysis, respectively, has revealed candidate 3-6 proteins and pathways altered by the 3-6 mutation. Candidates from both approaches will be validated. Specific Aim #3: To define the 3-6 induced changes in cellular lipid metabolism. We will determine how the 3-6 mutation alters the plasma membrane and/or ER lipid compositions. Sphingolipid trafficking will be examined in parental CHO cells and mutant 3-6. We will investigate the mechanism by which candidate 3-6 and proteins that suppress the phenotype modulate these membrane parameters.